title: New stabilized FastPrep-CLEAs for sialic acid synthesis.
abstract: N-acetyl-D-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-D-neuraminic acid (sialic acid) from N-acetyl-D-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K(M) for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.
authors.affiliation: Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus Espinardo, E-30100 Murcia, Spain.
authors.forename: María Inmaculada
authors.initials: MI
authors.lastname: García-García
authors.forename: Agustín
authors.initials: A
authors.lastname: Sola-Carvajal
authors.forename: Guiomar
authors.initials: G
authors.lastname: Sánchez-Carrón
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Carmona
authors.forename: Alvaro
authors.initials: A
authors.lastname: Sánchez-Ferrer
mesh_terms: D001709:Biotechnology; D004798:Enzymes / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000138:chemical synthesis*
chemical_list: D004798:Enzymes; D019158:N-Acetylneuraminic Acid